Open Access

5-HT2CR blockade in the amygdala conveys analgesic efficacy to SSRIs in a rat model of arthritis pain

Molecular Pain20139:41

https://doi.org/10.1186/1744-8069-9-41

Received: 28 June 2013

Accepted: 9 August 2013

Published: 12 August 2013

Abstract

Background

Pain, including arthritic pain, has a negative affective component and is often associated with anxiety and depression. However, selective serotonin reuptake inhibitor antidepressants (SSRIs) show limited effectiveness in pain. The amygdala plays a key role in the emotional-affective component of pain, pain modulation and affective disorders. Neuroplasticity in the basolateral and central amygdala (BLA and CeA, respectively) correlate positively with pain behaviors. Evidence suggests that serotonin receptor subtype 5-HT2CR in the amygdala contributes critically to anxiogenic behavior and anxiety disorders. In this study, we tested the hypothesis that 5-HT2CR in the amygdala accounts for the limited effectiveness of SSRIs in reducing pain behaviors and that 5-HT2CR blockade in the amygdala renders SSRIs effective.

Results

Nocifensive reflexes, vocalizations and anxiety-like behavior were measured in adult male Sprague–Dawley rats. Behavioral experiments were done in sham controls and in rats with arthritis induced by kaolin/carrageenan injections into one knee joint. Rats received a systemic (i.p.) administration of an SSRI (fluvoxamine, 30 mg/kg) or vehicle (sterile saline) and stereotaxic application of a selective 5-HT2CR antagonist (SB242084, 10 μM) or vehicle (ACSF) into BLA or CeA by microdialysis. Compared to shams, arthritic rats showed decreased hindlimb withdrawal thresholds (increased reflexes), increased duration of audible and ultrasonic vocalizations, and decreased open-arm choices in the elevated plus maze test suggesting anxiety-like behavior. Fluvoxamine (i.p.) or SB242084 (intra-BLA) alone had no significant effect, but their combination inhibited the pain-related increase of vocalizations and anxiety-like behavior without affecting spinal reflexes. SB242084 applied into the CeA in combination with systemic fluvoxamine had no effect on vocalizations and spinal reflexes.

Conclusions

The data suggest that 5-HT2CR in the amygdala, especially in the BLA, limits the effectiveness of SSRIs to inhibit pain-related emotional-affective behaviors.

Keywords

Amygdala Pain Serotonin SSRI 5-HT2CR Emotional-affective behavior Anxiety

Background

Pain is a multidimensional experience that includes not only sensory-discriminative but also emotional-affective and cognitive components [1, 2]. Certain antidepressants have become part of the therapeutic strategy for different types of persistent pain, including neuropathic pain, fibromyalgia, low back pain and headache [36], and they are also considered for osteoarthritis pain [7]. Selective serotonin reuptake inhibitor antidepressants (SSRIs) have low or inconsistent analgesic efficacy [4, 6] but better overall safety and tolerability compared to tricyclic antidepressants [8].

The serotonergic system has long been known to play an important role in pain modulation [9, 10]. The family of at least 14 serotonin (5-HT) receptor subtypes is divided into seven groups (5-HT1R – 5-HT7R) based on their structural and functional characteristics [1113]. The heterogeneity of 5-HT receptors is believed to account for the differential inhibitory or excitatory effects of 5-HT in the descending pain modulatory systems [9]. 5-HT2C receptor (5-HT2CR) has emerged as a major target for improved treatment of neuropsychiatric disorders such as anxiety disorders [1416]. 5-HT2CR has also been implicated in adverse effects of 5-HT and SSRIs [14] and in inconsistent clinical efficacy of SSRIs in neuropathic pain [17]. 5-HT2CR is a Gq/11 protein-coupled receptor expressed in GABAergic, glutamatergic, and dopaminergic neurons [18, 19]. Thus, 5-HT2CR can regulate the release of different transmitters to modulate excitatory and inhibitory neurotransmission [2022]. 5-HT2CR mRNA and protein show widespread distribution in the human and rat brain, including in the amygdala where particularly high levels are found in the lateral-basolateral area [23, 24].

The amygdala, a subcortical area known for its key role in emotions and affective disorders [25], is now recognized as an important neural substrate for the emotional-affective dimension of pain based on preclinical studies from our group [for reviews see [26, 27]] and others [2831] and clinical work [32, 33]. Amygdala activity correlates positively with pain behaviors in animals. Increases of amygdala activity can elicit or enhance pain responses even in the absence of tissue injury [3441]. Conversely, deactivation of the amygdala inhibits pain in different animal models [28, 4249]. Recent studies in humans also show increased amygdala activity in experimental and clinical pain [5054]. The amygdala circuitry that contributes to emotional-affective aspects of pain is centered on the lateral-basolateral (LA-BLA) and central (CeA) nuclei [26, 27]. The CeA receives nociceptive information through a direct pathway from the spinal cord and brainstem (external lateral parabrachial area) and highly processed affect-related information through an indirect pathway from the LA-BLA network via posterior thalamus [26, 27]. Neuroplasticity characterized by enhanced excitatory transmission [44, 45, 5561] and loss of inhibitory control [61, 62] develops in this circuitry in models of inflammatory and neuropathic pain. As a result, abnormally enhanced CeA output generates emotional-affective behaviors and modulates nocifensive responses through direct and indirect projections to brainstem and forebrain areas [26, 27].

The amygdala receives a strong serotonergic projection from the dorsal raphe nucleus [63, 64], which exerts excitatory and inhibitory effects on neuronal activity through different receptor subtypes [65, 66]. There is evidence for increased 5-HT release in the amygdala (BLA) in aversive states [16, 67, 68]. 5-HT2CR in the BLA but not CeA contributes critically to anxiogenic behavior and anxiety disorders [15, 16, 69] and mediates anxiogenic side effects of acutely administered antidepressants such as SSRIs [4, 70, 71]. Synaptic and cellular effects of 5-HT2CR in the amygdala are largely unknown but 5-HT2CR activation in the BLA facilitated NMDA receptor-mediated synaptic plasticity in BLA neurons [72] and induction of hippocampal LTP [73], suggesting that 5-HT2CR can control amygdala output.

In this study, we tested the hypothesis that 5-HT2CR in the amygdala (BLA but not CeA) contributes to the limited effectiveness of SSRIs on pain behaviors and that blockade of 5-HT2CR in the BLA renders SSRIs effective in reducing emotional-affective behaviors in a model of arthritis pain. To do so we applied a selective 5-HT2CR antagonist (SB242084 [74]) stereotaxically into BLA or CeA and administered a selective SSRI (fluvoxamine [75]) systemically (intraperitoneally, i.p.). The effect of each drug alone and of their combined application was determined.

Results

Spinal reflexes (hindlimb withdrawal thresholds), audible and ultrasonic vocalizations, and anxiety-like behavior in the elevated plus maze (EPM) were measured in adult male Sprague Dawley rats with (n = 61) or without (n = 38) arthritis. Arthritic pain was induced by intraarticular injections of kaolin and carrageen into the left knee joint. Sham rats were handled the same way as arthritic rats but the needle was inserted into the knee without injecting any compounds (see Methods). Behaviors and drug effects were measured 5–6 h after arthritis induction or needle insertion (shams). Drugs or vehicle were administered systemically (i.p.) and stereotaxically into the right basolateral (BLA) or central (CeA) nuclei of the amygdala (see Figure 1). Each animal was tested with only one drug regimen. Audible and ultrasonic vocalizations were measured simultaneously (see Methods) and spinal reflex thresholds were also determined in these animals (counter-balanced for order of tests). EPM performance was tested in separate groups of animals.
Figure 1

Histological verification of drug application sites. Diagrams adapted from Paxinos and Watson [107] show coronal sections through the right hemisphere at different levels posterior to bregma (indicated by numbers). Next to each diagram are shown in detail the basolateral (BLA) and central nuclei (CeA) of the amygdala. The boundaries of the different amygdala nuclei are easily identified under the microscope (see Figure 1 in [45]). Each symbol indicates the location of the tip of one microdialysis probe. Open circles, BLA; filled circles, CeA.

Co-application of intra-BLA SB242084 and systemic fluvoxamine decreased vocalizations and anxiety-like behavior of arthritic rats

Audible vocalizations

Audible vocalizations evoked by innocuous (300 g/30 mm2) and noxious (1200 g/30 mm2) compression of the knee for 15 s were measured in sham controls (Figure 2A and 2C) and in arthritic rats (Figure 2B and 2D). In sham rats, the following drug regimen had no effect on the duration of audible vocalizations compared to vehicle (n = 6 rats): stereotaxic application of a selective 5-HT2CR antagonist (SB242084 [74], 10 μM, concentration in microdialysis fiber) into BLA together with systemic vehicle administration (n = 7 rats); systemic administration of a selective SSRI (fluvoxamine [75], 30 mg/kg, i.p.) together with ACSF vehicle application into BLA (n = 7 rats); and combined application of systemic fluvoxamine and intra-BLA SB242084 (n = 7 rats). Arthritic rats showed increased audible vocalizations to normally innocuous (Figure 2B) and noxious (Figure 2D) stimuli, reflecting allodynia and hyperalgesia, respectively (see vehicle-treated group, n = 7 rats). Intra-BLA application of SB242084 (10 μM, n = 6) or systemic administration of fluvoxamine (30 mg/kg, i.p., n = 5) had no effect on audible vocalizations of arthritic rats. However, the combination of intra-BLA SB242084 and systemic fluvoxamine (n = 7) significantly decreased the duration of audible vocalizations to innocuous (P < 0.001) and noxious stimuli (P < 0.01, Dunnett’s multiple comparison tests), reversing the effect of arthritis. The data suggest that 5-HT2CR blockade allows an SSRI to inhibit higher integrated pain behaviors.
Figure 2

Drug effects on audible vocalizations. Effects of intra-BLA infusion of a selective 5-HT2CR antagonist (SB242084, 10 μM), systemic administration of an SSRI (fluvoxamine, 30 mg/kg, i.p) and combined administration of SB242084 and fluvoxamine on audible vocalizations evoked by innocuous (300 g/30 mm2, A,B) and noxious (1200 g/30 mm2, C,D) stimulation of the knee joint in sham control (A,C) and arthritic (B,D) rats. The drug combination had significant inhibitory effects in the arthritis pain model. *,*** P < 0.05, 0.001, compared to vehicle control (first bar in each histogram), one-way ANOVA followed by Dunnett’s multiple comparison tests. Bar histograms show means ± SEM of the total duration vocalizations over a 1 min period following the onset of the mechanical stimulus.

Ultrasonic vocalizations

Ultrasonic vocalizations evoked by innocuous (300 g/30 mm2) and noxious (1200 g/30 mm2) stimulation of the knee joint were measured in sham control (Figure 3A and 3C) and arthritic rats (Figure 3B and 3D). In sham rats, none of the drug regimen (SB242084, 10 μM, n = 7; fluvoxamine, 30 mg/kg, n = 7; co-application of SB242084 and fluvoxamine, n = 7 rats) had a significant effect on the duration of ultrasonic vocalizations compared to vehicle (n = 6 rats). Arthritic rats showed increased vocalizations to normally innocuous (Figure 3B) and noxious (Figure 3D) stimuli (see vehicle-treated group, n = 7 rats). The combination of intra-BLA SB242084 and systemic fluvoxamine (n = 7) inhibited ultrasonic vocalizations significantly (P < 0.001, Dunnett’s multiple comparison tests) compared to vehicle (n = 7 rats). SB242084 alone had no effect (n = 6) and fluvoxamine alone inhibited ultrasonic vocalizations to noxious stimuli slightly but significantly (n = 5, P < 0.05). The data suggest that 5-HT2CR blockade induces or enhances the ability of an SSRI to inhibit affective pain behaviors.
Figure 3

Drug effects on ultrasonic vocalizations. Effects of intra-BLA infusion of the selective 5-HT2CR antagonist (SB242084, 10 μM), systemic administration of the SSRI fluvoxamine (30 mg/kg, i.p) and combined administration of SB242084 and fluvoxamine on audible vocalizations evoked by innocuous (300 g/30 mm2, A,B) and noxious (1200 g/30 mm2, C,D) stimulation of the knee joint in sham control (A,C) and arthritic (B,D) rats. The drug combination had significant effects in the arthritis pain state. *,*** P < 0.05, 0.001, compared to vehicle control, one-way ANOVA followed by Dunnett’s multiple comparison tests. Same display as in Figure 2.

Anxiety-like behavior and locomotion

Open-arm choice in the elevated plus maze (EPM) was measured for 5 min in sham control rats and in arthritic rats as a negative indicator of anxiety-like behavior [76]. Compared to sham controls (n = 6), arthritic animals (n = 6) showed decreased preference for the open arms indicating an increase in anxiety-like behavior in the pain state (see vehicle control groups in Figure 4A and 4B). The combination of intra-BLA SB242084 (10 μM) and systemic fluvoxamine (30 mg/kg) had no effect in control rats (n = 5) compared to vehicle (n = 6) but increased the open-arm choice of arthritic rats (n = 6) compared to vehicle (n = 6) significantly (P < 0.05, Dunnett’s multiple comparison tests), suggesting an anxiolytic effect in the pain state. Intra-BLA SB242084 (n = 7) or systemic fluvoxamine (n = 7) alone had no significant effect in arthritic rats. Compared to sham controls (n = 6), arthritic rats (n = 6) showed decreased exploratory behaviour measured as the total number of entries into the open and closed arms of the EPM for 30 min (Figure 4C). None of the drug regimen (SB242084, 10 μM, n = 7; fluvoxamine, 30 mg/kg, n = 7; co-application of SB242084 and fluvoxamine, n = 6 rats) had a significant effect on locomotor activity in arthritic rats. In sham controls, only the combination of SB242084 and fluvoxamine was tested and had no significant effect on locomotor activity (n = 5).
Figure 4

Drug effects on anxiety-like behavior and locomotion. Open-arm choice in the elevated plus maze (EPM) was measured for 5 min as the ratio of open-arm entries to the total number of entries expressed as % in sham controls (A) and arthritic animals (B). The combination of intra-BLA SB242084 (10 μM) and systemic fluvoxamine (30 mg/kg) had no effect in sham controls but increased the open-arm choice of arthritic rats significantly. *P < 0.05, compared to vehicle, one-way ANOVA followed by Dunnett’s multiple comparison tests). (C) Evaluation of locomotor activity (total number of entries into the 4 arms of the EPM) in sham controls (left bar) and arthritic animals for 30 min. Locomotor activity was decreased in arthritic rats. The different drug regimen tested in arthritic rats had no effect. Bar histograms show means ± SEM.

Co-application of intra-BLA SB242084 and systemic fluvoxamine had no effect on spinal reflexes

Thresholds of hindlimb withdrawal reflexes evoked by mechanical compression of the knee joint were measured in sham controls (Figure 5A) and arthritic rats (Figure 5B). Compared to controls (n = 6 rats) arthritic rats (n = 7) had decreased thresholds, reflecting increased spinally organized reflexes and mechanical hypersensitivity (see vehicle groups in Figure 5A and 5B). None of the drug regimen had a significant effect in control rats (SB242084, 10 μM, n = 7; fluvoxamine, 30 mg/kg, n = 7; co-application of SB242084 and fluvoxamine, n = 7 rats) and in arthritic rats (SB242084, n = 6; fluvoxamine, n = 5; co-application of SB242084 and fluvoxamine, n = 7 rats). The data suggest that blockade of 5-HT2CR in the BLA allowed the serotonergic system to affect supraspinally but not spinally organized behaviors.
Figure 5

Drug effects on hindlimb withdrawal thresholds. Reflex thresholds were measured in sham controls (A) and arthritic rats (B) using a calibrated forceps for mechanical compression of the knee joint. Intra-BLA application of SB242084 (10 μM), systemic administration of fluvoxamine (30 mg/kg, i.p), and combined administration of SB242084 and fluvoxamine had no effect on reflex thresholds in control or arthritic rats. Bar histograms show means ± SEM.

Co-application of intra-CeA SB 242084 and systemic fluvoxamine had no effect on spinal reflexes and vocalizations in arthritic rats

Since the CeA serves as a major output nucleus for amygdala function related to pain modulation, we tested if 5-HT2CR also played a role in this nucleus (Figure 6). We only tested the combination of SB242084 and fluvoxamine since the previous results showed that fluvoxamine alone and SB242084 applied into BLA had no effect. We also performed these tests in arthritic animals only since the combination of intra-BLA SB242084 and systemic fluvoxamine had no effect in normal animals. The results show that coapplication of intra-CeA SB242084 (10 μM) and systemic fluvoxamine (30 mg/kg) had no significant effect on audible and ultrasonic vocalizations to (normally) innocuous and noxious stimuli and on spinal reflexes in arthritic rats (n = 5 rats for each parameter).
Figure 6

Drug application into CeA. Audible (A) and ultrasonic (B) vocalizations to innocuous and noxious compression of the knee (see Figure 2) and hindlimb withdrawal thresholds (C, see Figure 5) were measured in arthritic rats 5–6 h postinduction. Compared to vehicle controls the application of SB242084 (10 μM) into the CeA in combination with systemic administration of fluvoxamine (30 mg/kg, i.p) had no significant effect. Bar histograms show means ± SEM.

Discussion

The novelty of this study is the finding that blockade of 5-HT2CR in the BLA, but not CeA, can induce or enhance the ability of an SSRI to inhibit emotional-affective pain behaviors in an animal model of arthritis pain. The results suggest that 5-HT2CR in the BLA prohibits beneficial pain-inhibiting effects of serotonin (5-HT), which is consistent with previous reports that 5-HT2CR in the amygdala (BLA but not CeA) contributes critically to anxiogenic behavior and anxiety disorders [16, 69, 77] and mediates anxiogenic side effects of antidepressants including SSRIs [4, 70, 78]. 5-HT2CR antagonists have anxiolytic and antidepressant effects [7982], block the SSRI-induced increase of fear expression [71] and potentiate the antidepressant effects of SSRIs [83].

The present study provides not only further evidence for an important role of the amygdala in emotional-affective aspects of pain [26, 27] but also identifies the serotonergic system as a powerful modulator of amygdala function in pain. Blockade of 5-HT2CR by itself had no effect, but increasing the serotonergic drive with an SSRI engaged this receptor so that its blockade allowed 5-HT to inhibit pain behaviors. Since this strategy affected supraspinally organized behaviors (vocalizations and anxiety-like behaviors) but not spinal reflexes, the beneficial 5-HT effects appear to be due to an action in the brain. Our data suggest that 5-HT2CR acts in the BLA to block pain-inhibiting effects of SSRIs. The synaptic and cellular mechanisms remain to be determined.

The amygdala receives serotonergic input predominantly from the dorsal raphe nucleus [63, 64] and increased 5-HT release in the amygdala (BLA) is associated with aversive states [16, 67, 68]. 5-HT2CR activation in the BLA increased synaptic activation of BLA neurons [72]. A possibly scenario to explain the findings of our study is that 5-HT2CR activation drives BLA output to increase activity in the CeA, which serves as the output nucleus for major amygdala function and accounts for amygdala-dependent emotional-affective aspects of pain [26, 27]. 5-HT2CR knockout data linked 5-HT2CR-mediated anxiogenic behavior to the activation of CRF-containing neurons in the CeA [15]. Our previous studies established an important role of the CRF system in pain-related amygdala functions [35, 45, 8486]. CeA neurons project directly or indirectly to brainstem and forebrain areas involved in the expression of aversive behaviors and pain modulation, including the periaqueductal gray [26, 8789]. Direct brainstem projections from CeA can be glutamatergic [90], but CRF-containing CeA neurons also include a population of GABAergic neurons [91, 92]. Therefore, the positive correlation between amygdala output and pain behaviors can result from descending facilitation or disinhibition.

A single administration of fluvoxamine had pain-relieving effects in our study when combined with blockade of 5-HT2CR in the BLA, which is consistent with the observation that analgesic effects of anti-depressants are independent of their anti-depressant effects that usually occur only after weeks [5, 7]. Fluvoxamine alone had no effect except for a slight inhibition of ultrasonic vocalizations evoked by noxious stimulation in arthritic rats. Other studies reported weak effects of systemic fluvoxamine in the formalin pain test [93, 94] and mixed effects in the hot plate test [93, 95]. Intrathecal fluvoxamine had anti-allodynic effects in a neuropathic pain model (partial nerve ligation) and these were reduced by intrathecal administration of a 5-HT2A/2CR antagonist [96]. There is some evidence, however, that 5-HT2CR activation in the spinal cord may have inhibitory effects in neuropathic pain models [97, 98], possibly mediated by indirect noradrenergic mechanisms [99]. Our data suggest that 5-HT2CR activation in the amygdala mediates undesirable effects of 5-HT.

Some technical aspects of our study deserve consideration. We used selective compounds at concentrations that are well established in the literature (SB242084 [22, 100102]; fluvoxamine [75, 96]). However, while the drug concentration in the microdialysis fiber is known, the dose administered can only be estimated. Comparative data from our previous microdialysis and in vitro studies [45, 55, 58, 84, 85, 103, 104] indicate that the tissue concentration is at least 100 times lower than in the microdialysis probe due to the concentration gradient across the dialysis membrane and diffusion in the tissue. Therefore, drugs were dissolved in ACSF at a concentration 100 times that predicted to be needed. Microdialysis was chosen for drug delivery because it provides steady state drug levels without a volume effect [105]. Spread of drug and site of action need to be considered. Drug application into the CeA had no effect. These placement control experiments suggest that the drug did not spread beyond a distance of 1 mm around the tip of the microdialysis probe to reach the BLA, which is consistent with our previous estimates [35, 48, 84, 85]. The distance between the tips of the microdialysis probes in the BLA (effective drug administration site) and CeA (ineffective control site) is about 1 mm.

Conclusion

Pharmacological blockade of 5-HT2CR in the amygdala (BLA but not CeA) allows SSRIs to inhibit emotional-affective pain responses and anxiety-like behavior in an arthritis pain model. The study contributes novel insight into 5-HT functions in the brain and into brain mechanisms of pain.

Methods

Animals

Male Sprague Dawley rats (225–250 g) were housed in a temperature controlled room and maintained on a 12 h day/night cycle, with free access to food and water. On the day of the experiment, rats were transferred from the animal facility and allowed to acclimate to the laboratory for at least 1 h. At the end of the experiment, the animal was euthanized by decapitation using a guillotine (Harvard Apparatus Decapitator). All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Texas Medical Branch (UTMB) and conformed to the guidelines of the International Association for the Study of Pain (IASP) and of the National Institutes of Health (NIH).

Arthritis pain model and sham controls

A localized mono-arthritis was induced in the left knee joint by intra-articular injections of kaolin (4%, 80–100 μl) and carrageenan (2%, 80–100 μl) through the patellar ligament. This treatment paradigm reliably leads to inflammation and swelling of the knee within 1–3 h, reaches a maximum plateau at 5–6 h, and persists for several days [106]. Therefore, the 5–6 h time point was selected for measuring behaviors and testing drug effects. In sham control rats, the syringe was inserted into the knee joint cavity under the same conditions as in arthritic animals except that no compound was injected. Vehicle was not injected in sham animals because intraarticular saline injection causes a temporary swelling of the joint [55], which is one of the cardinal symptoms of an inflammation. To avoid any latent effect of increased intraarticular pressure, vehicle (sterile saline) was not injected in sham animals.

Experimental protocol

On Day 1, a guide cannula for drug (or artificial CSF, ACSF) application by microdialysis was stereotaxically inserted into the right BLA or CeA. On Day 2, 5–6 h after the induction of arthritis (or needle insertion for shams), behavioral experiments were performed 30 min after the systemic (i.p.) injection of the fluvoxamine or its vehicle (0.9% NaCl solution) in combination with an intra-BLA or intra-CeA application of SB242084 (or ACSF vehicle) for 20 min. SB242084 (selective 5-HT2CR antagonist) and fluvoxamine (SSRI) were purchased from Tocris Bioscience.

Drug application by microdialysis

Rats were deeply anaesthetized with pentobarbital sodium (Nembutal®, 50 mg/kg, i.p.) on Day 1. A guide cannula (David Kopf Instruments) was stereotaxically implanted into the right BLA or the right CeA, using the following coordinates: BLA, 2.8 mm caudal to bregma, 4.8 mm lateral to midline, 7.6 mm depth; CeA, 2.3 mm caudal to bregma, 4.0 mm lateral to midline, 7.0 mm depth [107]. Guide cannulas were affixed to the skull with dental acrylic (Plastic One, Roanoke, VA). Antibiotic ointment (Solosite Gel, Smith and Nephew) was applied to the exposed tissue to prevent infection. Local application of Lidocaine HCl (1%, 100 μl of 10 mg/ml) was done to minimize surgical pain and to prevent the animal from scratching of the surgical area upon recovery. On Day 2, a microdialysis probe (CMA/Microdialysis 11, Solna Sweden) that extended 1 mm beyond the tip of the guide cannula, was inserted for stereotaxic drug application into the amygdala. The probe was connected to an infusion pump (Harvard Apparatus, Holliston, MA) using polyethylene-50 tubing. Drugs or ACSF (vehicle) were applied for 20 min at a rate of 5 μl/min to establish equilibrium in the tissue. ACSF was oxygenated, equilibrated to pH 7.4 and contained the following (in mM): 125.0 NaCl, 2.6 KCl, 2.5 NaH2PO4, 1.3 CaCl2, 0.9 MgCl2, 21.0 NaHCO3, and 3.5 glucose. SB242084 was dissolved in ACSF on the day of the experiment at a concentration 100-fold that predicted to be needed in the tissue based on data in the literature [22, 100102] because of the concentration gradient across the dialysis membrane and diffusion in the tissue [45, 48, 84, 85]. At the end of the experiment, rats were decapitated and injection sites were verified histologically after injection of methylene blue (1 μl) and plotted on standard diagrams adapted from Paxinos and Watson [107] (see Figure 1).

Behavioral tests

Spinal reflexes

Thresholds of hindlimb withdrawal reflexes evoked by mechanical stimulation of the knee joint were measured as described in detail previously [106]. Mechanical stimuli of continuously increasing intensity were applied to the knee joint using a calibrated forceps equipped with a force transducer whose output was displayed (in g) on a screen. The area of tissue compressed by the tip of the forceps was 30 mm2. Withdrawal threshold was defined as the minimum stimulus intensity that evoked a withdrawal reflex. The test was repeated twice (5 min intervals) and the values were averaged to calculate the threshold (force in g/30 mm2).

Audible and ultrasonic vocalizations

Vocalizations were recorded and analyzed as described in detail previously [42]. The experimental setup (U.S. Patent 7,213,538) included a custom-designed recording chamber, a condenser microphone (20 Hz to 16 kHz) connected to a preamplifier, an ultrasound detector (25 ± 4 kHz), filter and amplifier (UltraVox 4-channel system; Noldus Information Technology, Leesburg, VA), and data acquisition software (UltraVox 2.0; Noldus Information Technology), which automatically monitored the occurrence of audible and ultrasonic vocalizations within user-defined frequencies and recorded number and duration of digitized events. Vocalizations in the audible and ultrasonic ranges were recorded simultaneously but with different microphones (condenser microphone and ultrasound detector, respectively) connected to separate channels of the amplifier. This computerized recording system was set to ignore sounds outside the defined frequency range. Animals were placed in the recording chamber for acclimation 1 h before the vocalization measurements. The recording chamber ensured the stable positioning of the animal at a fixed distance from the sound detectors and allowed the mechanical stimulation of the knee joint through openings for the hindlimbs. Brief (15 s) innocuous (300 g/30 mm2) and noxious (1200 g/30 mm2) mechanical stimuli were applied to the knee, using a calibrated forceps (see “Spinal reflexes”). Total duration of vocalizations (arithmetic sum of the duration of individual events) was recorded for 1 min, starting with the onset of the mechanical stimulus. Audible and ultrasonic vocalizations reflect supraspinally organized nocifensive and affective responses to aversive stimuli [106, 108].

Elevated plus maze test (EPM)

Anxiety-like behavior was determined using the EPM test as described previously [85, 106]. The EPM (Columbus Instruments, OH) was constructed from stainless steel to facilitate inter-trial cleaning for elimination of odor cues. A central quadrangle (10 × 10 cm) connected two opposing open arms (50 cm long, 10 cm wide) and two opposing closed arms (50 cm long, 10 cm wide, with 40 cm high walls on both sides), arranged in the shape of a plus. The platform was elevated 70 cm above the floor. An automated photocell system (Multi-Varimex v.1.00; Columbus Instruments, OH, USA) recorded movements of the animal on a personal computer. At the beginning of each trial, the animal was placed onto the central quadrangle facing an open-arm. The EPM was inside a dark enclosure to minimize anxiety levels in the absence of pain. Anxiety-like behavior was determined by measuring the open-arm preference (ratio of open-arm entries to the total number of entries expressed as %) for 5 min. Animals that stayed only in one arm were excluded from experiment.

Statistical analysis

All averaged values are given as the mean ± standard error of the mean (SEM). GraphPad Prism 3.0 software (Graph-Pad, San Diego, CA) was used for all statistical analysis. For multiple comparisons, one-way analysis of variance (ANOVA) was used followed by Dunnett’s multiple comparisons tests. Statistical significance was accepted at the level P < 0.05.

Authors’contributions

SG and VN conceptualized the hypothesis and designed the study. SG carried out the experiments, analyzed data, prepared figures and wrote the first draft of the manuscript. VN supervised the experiments, directed the data analysis, and finalized the manuscript. All authors read and approved the manuscript.

Abbreviations

5-HT: 

Serotonin

BLA: 

Basolateral nucleus of the amygdala

CeA: 

Central nucleus of the amygdala

LA: 

Lateral nucleus of the amygdala

SSRI: 

Selective serotonin reuptake inhibitor.

Declarations

Acknowledgements

This work was supported by National Institute of Neurological Disorders and Stroke Grants NS-081121, NS-38261 and NS-11255.

Authors’ Affiliations

(1)
Department of Neuroscience and Cell Biology, The University of Texas Medical Branch

References

  1. Auvray M, Myin E, Spence C: The sensory-discriminative and affective-motivational aspects of pain. Neurosci Biobehav Rev 2010, 34: 214–223. 10.1016/j.neubiorev.2008.07.008PubMedGoogle Scholar
  2. Moriarty O, McGuire BE, Finn DP: The effect of pain on cognitive function: a review of clinical and preclinical research. Prog Neurobiol 2011, 93: 385–404. 10.1016/j.pneurobio.2011.01.002PubMedGoogle Scholar
  3. Dharmshaktu P, Tayal V, Kalra BS: Efficacy of antidepressants as analgesics: a review. J Clin Pharmacol 2012, 52: 6–17. 10.1177/0091270010394852PubMedGoogle Scholar
  4. Lee YC, Chen PP: A review of SSRIs and SNRIs in neuropathic pain. Expert Opin Pharmacother 2010, 11: 2813–2825. 10.1517/14656566.2010.507192PubMedGoogle Scholar
  5. Perrot S, Javier RM, Marty M, Le JC, Laroche F: Is there any evidence to support the use of anti-depressants in painful rheumatological conditions? Systematic review of pharmacological and clinical studies. Rheumatology (Oxford) 2008, 47: 1117–1123. 10.1093/rheumatology/ken110Google Scholar
  6. Dworkin RH, O'Connor AB, Audette J, Baron R, Gourlay GK, Haanpaa ML, Kent JL, Krane EJ, LeBel AA, Levy RM, et al.: Recommendations for the pharmacological management of neuropathic pain: an overview and literature update. Mayo Clin Proc 2010, 85: S3-S14.PubMed CentralPubMedGoogle Scholar
  7. Citrome L, Weiss-Citrome A: Antidepressants and the relief of osteoarthritic pain - findings from a study examining adjunctive duloxetine. Int J Clin Pract 2012, 66: 431–433. 10.1111/j.1742-1241.2012.02899.xPubMedGoogle Scholar
  8. Ferguson JM: SSRI Antidepressant medications: Adverse effects and tolerability. Prim Care Companion J Clin Psychiatry 2001, 3: 22–27. 10.4088/PCC.v03n0105PubMed CentralPubMedGoogle Scholar
  9. Ossipov MH, Dussor GO, Porreca F: Central modulation of pain. J Clin Invest 2010, 120: 3779–3787. 10.1172/JCI43766PubMed CentralPubMedGoogle Scholar
  10. Fields HL, Heinricher MM, Mason P: Neurotransmitters in nociceptive modulatory circuits. Annu Rev Neurosci 1991, 14: 219–245. 10.1146/annurev.ne.14.030191.001251PubMedGoogle Scholar
  11. Millan MJ, Marin P, Bockaert J, la Cour CM: Signaling at G-protein-coupled serotonin receptors: recent advances and future research directions. Trends Pharmacol Sci 2008, 29: 454–464. 10.1016/j.tips.2008.06.007PubMedGoogle Scholar
  12. Bockaert J, Claeysen S, Becamel C, Dumuis A, Marin P: Neuronal 5-HT metabotropic receptors: fine-tuning of their structure, signaling, and roles in synaptic modulation. Cell Tissue Res 2006, 326: 553–572. 10.1007/s00441-006-0286-1PubMedGoogle Scholar
  13. Hannon J, Hoyer D: Molecular biology of 5-HT receptors. Behav Brain Res 2008, 195: 198–213. 10.1016/j.bbr.2008.03.020PubMedGoogle Scholar
  14. Jensen NH, Cremers TI, Sotty F: Therapeutic potential of 5-HT2C receptor ligands. Scientific World Journal 2010, 10: 1870–1885.PubMedGoogle Scholar
  15. Heisler LK, Zhou L, Bajwa P, Hsu J, Tecott LH: Serotonin 5-HT(2C) receptors regulate anxiety-like behavior. Genes Brain Behav 2007, 6: 491–496. 10.1111/j.1601-183X.2007.00316.xPubMedGoogle Scholar
  16. Christianson JP, Ragole T, Amat J, Greenwood BN, Strong PV, Paul ED, Fleshner M, Watkins LR, Maier SF: 5-hydroxytryptamine 2C receptors in the basolateral amygdala are involved in the expression of anxiety after uncontrollable traumatic stress. Biol Psychiatry 2010, 67: 339–345. 10.1016/j.biopsych.2009.09.011PubMed CentralPubMedGoogle Scholar
  17. Brasch-Andersen C, Moller MU, Christiansen L, Thinggaard M, Otto M, Brosen K, Sindrup SH: A candidate gene study of serotonergic pathway genes and pain relief during treatment with escitalopram in patients with neuropathic pain shows significant association to serotonin receptor2C (HTR2C). Eur J Clin Pharmacol 2011, 67: 1131–1137. 10.1007/s00228-011-1056-xPubMedGoogle Scholar
  18. Liu S, Bubar MJ, Lanfranco MF, Hillman GR, Cunningham KA: Serotonin(2C) receptor localization in GABA neurons of the rat medial prefrontal cortex: Implications for understanding the neurobiology of addiction. Neuroscience 2007, 146: 1667–1688.Google Scholar
  19. Bubar MJ, Stutz SJ, Cunningham KA: 5-HT(2C) receptors localize to dopamine and GABA neurons in the rat mesoaccumbens pathway. PLoS One 2011, 6: e20508. 10.1371/journal.pone.0020508PubMed CentralPubMedGoogle Scholar
  20. Tardito D, Milanese M, Bonifacino T, Musazzi L, Grilli M, Mallei A, Mocaer E, Gabriel-Gracia C, Racagni G, Popoli M, et al.: Blockade of stress-induced increase of glutamate release in the rat prefrontal/frontal cortex by agomelatine involves synergy between melatonergic and 5-HT2C receptor-dependent pathways. BMC Neurosci 2010, 11: 68. 10.1186/1471-2202-11-68PubMed CentralPubMedGoogle Scholar
  21. Theile JW, Morikawa H, Gonzales RA, Morrisett RA: Role of 5-hydroxytryptamine2C receptors in Ca2+−dependent ethanol potentiation of GABA release onto ventral tegmental area dopamine neurons. J Pharmacol Exp Ther 2009, 329: 625–633. 10.1124/jpet.108.147793PubMed CentralPubMedGoogle Scholar
  22. Navailles S, Moison D, Cunningham KA, Spampinato U: Differential regulation of the mesoaccumbens dopamine circuit by serotonin2C receptors in the ventral tegmental area and the nucleus accumbens: an in vivo microdialysis study with cocaine. Neuropsychopharmacology 2008, 33: 237–246. 10.1038/sj.npp.1301414PubMedGoogle Scholar
  23. Clemett DA, Punhani T, Duxon MS, Blackburn TP, Fone KC: Immunohistochemical localisation of the 5-HT2C receptor protein in the rat CNS. Neuropharmacology 2000, 39: 123–132. 10.1016/S0028-3908(99)00086-6PubMedGoogle Scholar
  24. Pompeiano M, Palacios JM, Mengod G: Distribution of the serotonin 5-HT 2 receptor family mRNAs: Comparison between 5-HT 2A and 5-HT 2C receptors. Mol Brain Res 1994, 23: 163–178. 10.1016/0169-328X(94)90223-2PubMedGoogle Scholar
  25. Phelps EA, Ledoux JE: Contributions of the Amygdala to Emotion Processing: From Animal Models to Human Behavior. Neuron 2005, 48: 175–187. 10.1016/j.neuron.2005.09.025PubMedGoogle Scholar
  26. Neugebauer V, Li W, Bird GC, Han JS: The amygdala and persistent pain. Neuroscientist 2004, 10: 221–234. 10.1177/1073858403261077PubMedGoogle Scholar
  27. Neugebauer V, Galhardo V, Maione S, Mackey SC: Forebrain pain mechanisms. Brain Res Rev 2009, 60: 226–242. 10.1016/j.brainresrev.2008.12.014PubMed CentralPubMedGoogle Scholar
  28. Pedersen LH, Scheel-Kruger J, Blackburn-Munro G: Amygdala GABA-A receptor involvement in mediating sensory-discriminative and affective-motivational pain responses in a rat model of peripheral nerve injury. Pain 2007, 127: 17–26. 10.1016/j.pain.2006.06.036PubMedGoogle Scholar
  29. Myers B, Greenwood-Van Meerveld B: Corticosteroid receptor-mediated mechanisms in the amygdala regulate anxiety and colonic sensitivity. Am J Physiol Gastrointest Liver Physiol 2007, 292: G1622-G1629. 10.1152/ajpgi.00080.2007PubMedGoogle Scholar
  30. Ansah OB, Bourbia N, Goncalves L, Almeida A, Pertovaara A: Influence of amygdaloid glutamatergic receptors on sensory and emotional pain-related behavior in the neuropathic rat. Behav Brain Res 2010, 209: 174–178. 10.1016/j.bbr.2010.01.021PubMedGoogle Scholar
  31. Tanimoto S, Nakagawa T, Yamauchi Y, Minami M, Satoh M: Differential contributions of the basolateral and central nuclei of the amygdala in the negative affective component of chemical somatic and visceral pains in rats. Eur J Neurosci 2003, 18: 2343–2350. 10.1046/j.1460-9568.2003.02952.xPubMedGoogle Scholar
  32. Borsook D, Sava S, Becerra L: The pain imaging revolution: advancing pain into the 21st century. Neuroscientist 2010, 16: 171–185. 10.1177/1073858409349902PubMed CentralPubMedGoogle Scholar
  33. Apkarian AV, Hashmi JA, Baliki MN: Pain and the brain: specificity and plasticity of the brain in clinical chronic pain. Pain 2011, 152: S49-S64. 10.1016/j.pain.2010.11.010PubMed CentralPubMedGoogle Scholar
  34. Han JS, Adwanikar H, Li Z, Ji G, Neugebauer V: Facilitation of synaptic transmission and pain responses by CGRP in the amygdala of normal rats. Mol Pain 2010, 6: 10–23. 10.1186/1744-8069-6-10PubMed CentralPubMedGoogle Scholar
  35. Ji G, Fu Y, Adwanikar H, Neugebauer V: Non-pain-related CRF1 activation in the amygdala facilitates synaptic transmission and pain responses. Mol Pain 2013, 9: 2. 10.1186/1744-8069-9-2PubMed CentralPubMedGoogle Scholar
  36. Carrasquillo Y, Gereau RW: Activation of the extracellular signal-regulated kinase in the amygdala modulates pain perception. J Neurosci 2007, 27: 1543–1551. 10.1523/JNEUROSCI.3536-06.2007PubMedGoogle Scholar
  37. Kolber BJ, Montana MC, Carrasquillo Y, Xu J, Heinemann SF, Muglia LJ, Gereau RW: Activation of metabotropic glutamate receptor 5 in the amygdala modulates pain-like behavior. J Neurosci 2010, 30: 8203–8213. 10.1523/JNEUROSCI.1216-10.2010PubMed CentralPubMedGoogle Scholar
  38. Qin C, Greenwood-Van Meerveld B, Foreman RD: Visceromotor and spinal neuronal responses to colorectal distension in rats with aldosterone onto the amygdala. J Neurophysiol 2003, 90: 2–11. 10.1152/jn.00023.2003PubMedGoogle Scholar
  39. Myers DA, Gibson M, Schulkin J, Greenwood Van-Meerveld B: Corticosterone implants to the amygdala and type 1 CRH receptor regulation: effects on behavior and colonic sensitivity. Behav Brain Res 2005, 161: 39–44. 10.1016/j.bbr.2005.03.001PubMedGoogle Scholar
  40. Myers B, Greenwood-Van MB: Divergent effects of amygdala glucocorticoid and mineralocorticoid receptors in the regulation of visceral and somatic pain. Am J Physiol Gastrointest Liver Physiol 2010, 298: G295-G303. 10.1152/ajpgi.00298.2009PubMedGoogle Scholar
  41. Li Z, Ji G, Neugebauer V: Mitochondrial reactive oxygen species are activated by mGluR5 through IP3 and activate ERK and PKA to increase excitability of amygdala neurons and pain behavior. J Neurosci 2011, 31: 1114–1127. 10.1523/JNEUROSCI.5387-10.2011PubMed CentralPubMedGoogle Scholar
  42. Han JS, Neugebauer V: mGluR1 and mGluR5 antagonists in the amygdala inhibit different components of audible and ultrasonic vocalizations in a model of arthritic pain. Pain 2005, 113: 211–222. 10.1016/j.pain.2004.10.022PubMedGoogle Scholar
  43. Martin TJ, Buechler NL, Kim SA, Ewan EE, Xiao R, Childers SR: Involvement of the lateral amygdala in the antiallodynic and reinforcing effects of heroin in rats after peripheral nerve injury. Anesthesiology 2011, 114: 633–642. 10.1097/ALN.0b013e318209aba7PubMed CentralPubMedGoogle Scholar
  44. Han JS, Li W, Neugebauer V: Critical role of calcitonin gene-related peptide 1 receptors in the amygdala in synaptic plasticity and pain behavior. J Neurosci 2005, 25: 10717–10728. 10.1523/JNEUROSCI.4112-05.2005PubMedGoogle Scholar
  45. Fu Y, Neugebauer V: Differential mechanisms of CRF1 and CRF2 receptor functions in the amygdala in pain-related synaptic facilitation and behavior. J Neurosci 2008, 28: 3861–3876. 10.1523/JNEUROSCI.0227-08.2008PubMed CentralPubMedGoogle Scholar
  46. Fu Y, Han J, Ishola T, Scerbo M, Adwanikar H, Ramsey C, Neugebauer V: PKA and ERK, but not PKC, in the amygdala contribute to pain-related synaptic plasticity and behavior. Mol Pain 2008, 4: 26–46. 10.1186/1744-8069-4-26PubMed CentralPubMedGoogle Scholar
  47. Palazzo E, Fu Y, Ji G, Maione S, Neugebauer V: Group III mGluR7 and mGluR8 in the amygdala differentially modulate nocifensive and affective pain behaviors. Neuropharmacology 2008, 55: 537–545. 10.1016/j.neuropharm.2008.05.007PubMed CentralPubMedGoogle Scholar
  48. Ji G, Sun H, Fu Y, Li Z, Pais-Vieira M, Galhardo V, Neugebauer V: Cognitive impairment in pain through amygdala-driven prefrontal cortical deactivation. J Neurosci 2010, 30: 5451–5464. 10.1523/JNEUROSCI.0225-10.2010PubMed CentralPubMedGoogle Scholar
  49. Hebert MA, Ardid D, Henrie JA, Tamashiro K, Blanchard DC, Blanchard RJ: Amygdala lesions produce analgesia in a novel, ethologically relevant acute pain test. Phys and Behav 1999, 67: 99–105. 10.1016/S0031-9384(99)00042-6Google Scholar
  50. Liu CC, Ohara S, Franaszczuk P, Zagzoog N, Gallagher M, Lenz FA: Painful stimuli evoke potentials recorded from the medial temporal lobe in humans. Neuroscience 2010, 165: 1402–1411. 10.1016/j.neuroscience.2009.11.026PubMed CentralPubMedGoogle Scholar
  51. Baliki MN, Geha PY, Jabakhanji R, Harden N, Schnitzer TJ, Apkarian AV: A preliminary fMRI study of analgesic treatment in chronic back pain and knee osteoarthritis. Mol Pain 2008, 4: 47. 10.1186/1744-8069-4-47PubMed CentralPubMedGoogle Scholar
  52. Tillisch K, Mayer EA, Labus JS: Quantitative Meta-Analysis Identifies Brain Regions Activated During Rectal Distension in Irritable Bowel Syndrome. Gastroenterology 2010, 140: 91–100.PubMed CentralPubMedGoogle Scholar
  53. Kulkarni B, Bentley DE, Elliott R, Julyan PJ, Boger E, Watson A, Boyle Y, El-Deredy W, Jones AK: Arthritic pain is processed in brain areas concerned with emotions and fear. Arthritis Rheum 2007, 56: 1345–1354. 10.1002/art.22460PubMedGoogle Scholar
  54. Simons LE, Moulton EA, Linnman C, Carpino E, Becerra L, Borsook D: The human amygdala and pain: Evidence from neuroimaging. Hum Brain Mapp 2012. PMID: 23097300 10.1002/hbm.22199Google Scholar
  55. Neugebauer V, Li W, Bird GC, Bhave G, Gereau RW: Synaptic plasticity in the amygdala in a model of arthritic pain: differential roles of metabotropic glutamate receptors 1 and 5. J Neurosci 2003, 23: 52–63.PubMedGoogle Scholar
  56. Adedoyin MO, Vicini S, Neale JH: Endogenous N-acetylaspartylglutamate (NAAG) inhibits synaptic plasticity/transmission in the amygdala in a mouse inflammatory pain model. Mol Pain 2010, 6: 60–77.PubMed CentralPubMedGoogle Scholar
  57. Han JS, Neugebauer V: Synaptic plasticity in the amygdala in a visceral pain model in rats. Neurosci Lett 2004, 361: 254–257. 10.1016/j.neulet.2003.12.027PubMedGoogle Scholar
  58. Bird GC, Lash LL, Han JS, Zou X, Willis WD, Neugebauer V: Protein kinase A-dependent enhanced NMDA receptor function in pain-related synaptic plasticity in rat amygdala neurones. J Physiol 2005, 564: 907–921. 10.1113/jphysiol.2005.084780PubMed CentralPubMedGoogle Scholar
  59. Ikeda R, Takahashi Y, Inoue K, Kato F: NMDA receptor-independent synaptic plasticity in the central amygdala in the rat model of neuropathic pain. Pain 2007, 127: 161–172. 10.1016/j.pain.2006.09.003PubMedGoogle Scholar
  60. Nakao A, Takahashi Y, Nagase M, Ikeda R, Kato F: Role of capsaicin-sensitive C-fiber afferents in neuropathic pain-induced synaptic potentiation in the nociceptive amygdala. Mol Pain 2012, 8: 51. 10.1186/1744-8069-8-51PubMed CentralPubMedGoogle Scholar
  61. Ren W, Neugebauer V: Pain-related increase of excitatory transmission and decrease of inhibitory transmission in the central nucleus of the amygdala are mediated by mGluR1. Mol Pain 2010, 6: 93–106. 10.1186/1744-8069-6-93PubMed CentralPubMedGoogle Scholar
  62. Ren W, Palazzo E, Maione S, Neugebauer V: Differential effects of mGluR7 and mGluR8 activation on pain-related synaptic activity in the amygdala. Neuropharmacology 2011, 61: 1334–1344. 10.1016/j.neuropharm.2011.08.006PubMed CentralPubMedGoogle Scholar
  63. Ma QP, Yin GF, Ai MK, Han JS: Serotonergic projections from the nucleus raphe dorsalis to the amygdala in the rat. Neurosci Lett 1991, 134: 21–24. 10.1016/0304-3940(91)90499-JPubMedGoogle Scholar
  64. Lowry CA: Functional subsets of serotonergic neurones: implications for control of the hypothalamic-pituitary-adrenal axis. J Neuroendocrinol 2002, 14: 911–923. 10.1046/j.1365-2826.2002.00861.xPubMedGoogle Scholar
  65. Stein C, Davidowa H, Albrecht D: 5-HT(1A) receptor-mediated inhibition and 5-HT(2) as well as 5-HT(3) receptor-mediated excitation in different subdivisions of the rat amygdala. Synapse 2000, 38: 328–337. 10.1002/1098-2396(20001201)38:3<328::AID-SYN12>3.0.CO;2-TPubMedGoogle Scholar
  66. Rainnie DG: Serotonergic modulation of neurotransmission in the rat basolateral amygdala. J Neurophysiol 1999, 82: 69–85.PubMedGoogle Scholar
  67. Funada M, Hara C: Differential effects of psychological stress on activation of the 5-hydroxytryptamine- and dopamine-containing neurons in the brain of freely moving rats. Brain Res 2001, 901: 247–251. 10.1016/S0006-8993(01)02160-6PubMedGoogle Scholar
  68. Macedo CE, Martinez RCR, de Souza Silva MA, Brandao ML: Increases in extracellular levels of 5-HT and dopamine in the basolateral, but not in the central, nucleus of amygdala induced by aversive stimulation of the inferior colliculus. Eur J Neurosci 2005, 21: 1131–1138. 10.1111/j.1460-9568.2005.03939.xPubMedGoogle Scholar
  69. Campbell BM, Merchant KM: Serotonin 2C receptors within the basolateral amygdala induce acute fear-like responses in an open-field environment. Brain Res 2003, 993: 1–9. 10.1016/S0006-8993(03)03384-5PubMedGoogle Scholar
  70. Ravinder S, Pillai AG, Chattarji S: Cellular correlates of enhanced anxiety caused by acute treatment with the selective serotonin reuptake inhibitor fluoxetine in rats. Front Behav Neurosci 2011, 5: 88.PubMed CentralPubMedGoogle Scholar
  71. Burghardt NS, Bush DE, McEwen BS, LeDoux JE: Acute selective serotonin reuptake inhibitors increase conditioned fear expression: blockade with a 5-HT(2C) receptor antagonist. Biol Psychiatry 2007, 62: 1111–1118. 10.1016/j.biopsych.2006.11.023PubMed CentralPubMedGoogle Scholar
  72. Chen A, Hough CJ, Li H: Serotonin type II receptor activation facilitates synaptic plasticity via N-methyl-D-aspartate-mediated mechanism in the rat basolateral amygdala. Neuroscience 2003, 119: 53–63. 10.1016/S0306-4522(03)00076-9PubMedGoogle Scholar
  73. Abe K, Fujimoto T, Akaishi T, Misawa M: Stimulation of basolateral amygdaloid serotonin 5-HT(2C) receptors promotes the induction of long-term potentiation in the dentate gyrus of anesthetized rats. Neurosci Lett 2009, 451: 65–68. 10.1016/j.neulet.2008.12.023PubMedGoogle Scholar
  74. Kennett GA, Wood MD, Bright F, Trail B, Riley G, Holland V, Avenell KY, Stean T, Upton N, Bromidge S, et al.: SB 242084, a selective and brain penetrant 5-HT2C receptor antagonist. Neuropharmacology 1997, 36: 609–620. 10.1016/S0028-3908(97)00038-5PubMedGoogle Scholar
  75. Nakae A, Nakai K, Tanaka T, Hagihira S, Shibata M, Ueda K, Masimo T: The role of RNA editing of the serotonin 2C receptor in a rat model of oro-facial neuropathic pain. Eur J Neurosci 2008, 27: 2373–2379. 10.1111/j.1460-9568.2008.06205.xPubMedGoogle Scholar
  76. Walf AA, Frye CA: The use of the elevated plus maze as an assay of anxiety-related behavior in rodents. Nat Protoc 2007, 2: 322–328. 10.1038/nprot.2007.44PubMed CentralPubMedGoogle Scholar
  77. Griebel G, Perrault G, Sanger DJ: A comparative study of the effects of selective and non-selective 5-HT2 receptor subtype antagonists in rat and mouse models of anxiety. Neuropharmacology 1997, 36: 793–802. 10.1016/S0028-3908(97)00034-8PubMedGoogle Scholar
  78. Vicente MA, Zangrossi H: Serotonin-2C receptors in the basolateral nucleus of the amygdala mediate the anxiogenic effect of acute imipramine and fluoxetine administration. Int J Neuropsychopharmacol 2012, 14: 389–400.Google Scholar
  79. Dekeyne A, la Mannoury CC, Gobert A, Brocco M, Lejeune F, Serres F, Sharp T, Daszuta A, Soumier A, Papp M, et al.: S32006, a novel 5-HT2C receptor antagonist displaying broad-based antidepressant and anxiolytic properties in rodent models. Psychopharmacology (Berl) 2008, 199: 549–568. 10.1007/s00213-008-1177-9Google Scholar
  80. Harada K, Aota M, Inoue T, Matsuda R, Mihara T, Yamaji T, Ishibashi K, Matsuoka N: Anxiolytic activity of a novel potent serotonin 5-HT2C receptor antagonist FR260010: a comparison with diazepam and buspirone. Eur J Pharmacol 2006, 553: 171–184. 10.1016/j.ejphar.2006.09.042PubMedGoogle Scholar
  81. Kantor S, Jakus R, Molnar E, Gyongyosi N, Toth A, Detari L, Bagdy G: Despite similar anxiolytic potential, the 5-hydroxytryptamine 2C receptor antagonist SB-242084 [6-chloro-5-methyl-1-[2-(2-methylpyrid-3-yloxy)-pyrid-5-yl carbamoyl] indoline] and chlordiazepoxide produced differential effects on electroencephalogram power spectra. J Pharmacol Exp Ther 2005, 315: 921–930. 10.1124/jpet.105.086413PubMedGoogle Scholar
  82. Dekeyne A, Brocco M, Loiseau F, Gobert A, Rivet JM, Di CB, Cremers TI, Flik G, Fone KC, Watson DJ, et al.: S32212, a novel serotonin type 2C receptor inverse agonist/alpha2-adrenoceptor antagonist and potential antidepressant: II. A behavioral, neurochemical, and electrophysiological characterization. J Pharmacol Exp Ther 2012, 340: 765–780. 10.1124/jpet.111.187534PubMedGoogle Scholar
  83. Cremers TI, Giorgetti M, Bosker FJ, Hogg S, Arnt J, Mork A, Honig G, Bogeso KP, Westerink BH, den BH, et al.: Inactivation of 5-HT(2C) receptors potentiates consequences of serotonin reuptake blockade. Neuropsychopharmacology 2004, 29: 1782–1789. 10.1038/sj.npp.1300474PubMedGoogle Scholar
  84. Ji G, Neugebauer V: Differential effects of CRF1 and CRF2 receptor antagonists on pain-related sensitization of neurons in the central nucleus of the amygdala. J Neurophysiol 2007, 97: 3893–3904. 10.1152/jn.00135.2007PubMedGoogle Scholar
  85. Ji G, Fu Y, Ruppert KA, Neugebauer V: Pain-related anxiety-like behavior requires CRF1 receptors in the amygdala. Mol Pain 2007, 3: 13–17. 10.1186/1744-8069-3-13PubMed CentralPubMedGoogle Scholar
  86. Ji G, Neugebauer V: Pro- and anti-nociceptive effects of corticotropin-releasing factor (CRF) in Central Amygdala neurons are mediated through different receptors. J Neurophysiol 2008, 99: 1201–1212. 10.1152/jn.01148.2007PubMedGoogle Scholar
  87. Schiess MC, Callahan PM, Zheng H: Characterization of the electrophysiological and morphological properties of rat central amygdala neurons in vitro. J Neurosci Res 1999, 58: 663–673. 10.1002/(SICI)1097-4547(19991201)58:5<663::AID-JNR7>3.0.CO;2-APubMedGoogle Scholar
  88. Bourgeais L, Gauriau C, Bernard J-F: Projections from the nociceptive area of the central nucleus of the amygdala to the forebrain: a PHA-L study in the rat. Eur J Neurosci 2001, 14: 229–255. 10.1046/j.0953-816x.2001.01640.xPubMedGoogle Scholar
  89. Gray TS, Magnuson DJ: Peptide immunoreactive neurons in the amygdala and the bed nucleus of the stria terminalis project to the midbrain central gray in the rat. Peptides 1992, 13: 451–460. 10.1016/0196-9781(92)90074-DPubMedGoogle Scholar
  90. Lee HS, Kim MA, Valentino RJ, Waterhouse BD: Glutamatergic afferent projections to the dorsal raphe nucleus of the rat. Brain Res 2003, 963: 57–71. 10.1016/S0006-8993(02)03841-6PubMedGoogle Scholar
  91. Veinante P, Stoeckel ME, Freund-Mercier MJ: GABA- and peptide-immunoreactivities co-localize in the rat central extended amygdala. Neurorep 1997, 8: 2985–2989. 10.1097/00001756-199709080-00035Google Scholar
  92. Day HE, Curran EJ, Watson SJ Jr, Akil H: Distinct neurochemical populations in the rat central nucleus of the amygdala and bed nucleus of the stria terminalis: evidence for their selective activation by interleukin-1beta. J Comp Neurol 1999, 413: 113–128. 10.1002/(SICI)1096-9861(19991011)413:1<113::AID-CNE8>3.0.CO;2-BPubMedGoogle Scholar
  93. Otsuka N, Kiuchi Y, Yokogawa F, Masuda Y, Oguchi K, Hosoyamada A: Antinociceptive efficacy of antidepressants: assessment of five antidepressants and four monoamine receptors in rats. J Anesth 2001, 15: 154–158. 10.1007/s005400170018PubMedGoogle Scholar
  94. Yokogawa F, Kiuchi Y, Ishikawa Y, Otsuka N, Masuda Y, Oguchi K, Hosoyamada A: An investigation of monoamine receptors involved in antinociceptive effects of antidepressants. Anesth Analg 2002, 95: 163–168. table 10.1097/00000539-200207000-00029PubMedGoogle Scholar
  95. Schreiber S, Backer MM, Yanai J, Pick CG: The antinociceptive effect of fluvoxamine. Eur Neuropsychopharmacol 1996, 6: 281–284. 10.1016/S0924-977X(96)00031-4PubMedGoogle Scholar
  96. Honda M, Uchida K, Tanabe M, Ono H: Fluvoxamine, a selective serotonin reuptake inhibitor, exerts its antiallodynic effects on neuropathic pain in mice via 5-HT2A/2C receptors. Neuropharmacology 2006, 51: 866–872. 10.1016/j.neuropharm.2006.05.031PubMedGoogle Scholar
  97. Nakai K, Nakae A, Oba S, Mashimo T, Ueda K: 5-HT2C receptor agonists attenuate pain-related behaviour in a rat model of trigeminal neuropathic pain. Eur J Pain 2010, 14: 999–1006. 10.1016/j.ejpain.2010.04.008PubMedGoogle Scholar
  98. Obata H, Saito S, Sakurazawa S, Sasaki M, Usui T, Goto F: Antiallodynic effects of intrathecally administered 5-HT(2C) receptor agonists in rats with nerve injury. Pain 2004, 108: 163–169. 10.1016/j.pain.2003.12.019PubMedGoogle Scholar
  99. Obata H, Ito N, Sasaki M, Saito S, Goto F: Possible involvement of spinal noradrenergic mechanisms in the antiallodynic effect of intrathecally administered 5-HT2C receptor agonists in the rats with peripheral nerve injury. Eur J Pharmacol 2007, 567: 89–94. 10.1016/j.ejphar.2007.03.029PubMedGoogle Scholar
  100. Liu S, Cunningham KA: Serotonin(2C) receptors (5-HT(2C)R) control expression of cocaine-induced conditioned hyperactivity. Drug Alcohol Depend 2006, 81: 275–282. 10.1016/j.drugalcdep.2005.07.007PubMedGoogle Scholar
  101. Zayara AE, McIver G, Valdivia PN, Lominac KD, McCreary AC, Szumlinski KK: Blockade of nucleus accumbens 5-HT2A and 5-HT2C receptors prevents the expression of cocaine-induced behavioral and neurochemical sensitization in rats. Psychopharmacology (Berl) 2011, 213: 321–335. 10.1007/s00213-010-1996-3Google Scholar
  102. Calcagno E, Invernizzi RW: Strain-dependent serotonin neuron feedback control: role of serotonin 2C receptors. J Neurochem 2010, 114: 1701–1710. 10.1111/j.1471-4159.2010.06880.xPubMedGoogle Scholar
  103. Li W, Neugebauer V: Differential roles of mGluR1 and mGluR5 in brief and prolonged nociceptive processing in central amygdala neurons. J Neurophysiol 2004, 91: 13–24.PubMedGoogle Scholar
  104. Li W, Neugebauer V: Block of NMDA and non-NMDA receptor activation results in reduced background and evoked activity of central amygdala neurons in a model of arthritic pain. Pain 2004, 110: 112–122. 10.1016/j.pain.2004.03.015PubMedGoogle Scholar
  105. Stiller CO, Taylor BK, Linderoth B, Gustafsson H, Warsame Afrah A, Brodin E: Microdialysis in pain research. Adv Drug Deliv Rev 2003, 55: 1065–1079. 10.1016/S0169-409X(03)00104-2PubMedGoogle Scholar
  106. Neugebauer V, Han JS, Adwanikar H, Fu Y, Ji G: Techniques for assessing knee joint pain in arthritis. Mol Pain 2007, 3: 8–20. 10.1186/1744-8069-3-8PubMed CentralPubMedGoogle Scholar
  107. Paxinos G, Watson C: The rat brain in stereotaxic coordinates. New York: Academic Press; 1998.Google Scholar
  108. Borszcz GS, Leaton RN: The effect of amygdala lesions on conditional and unconditional vocalizations in rats. Neurobiol Learn and Mem 2003, 79: 212–225. 10.1016/S1074-7427(03)00002-9Google Scholar

Copyright

© Grégoire and Neugebauer; licensee BioMed Central Ltd. 2013

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.